Expanding the Dipeptidyl Peptidase 4-Regulated Peptidome via an Optimized Peptidomics Platform

Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, USA.
Journal of the American Chemical Society (Impact Factor: 12.11). 02/2010; 132(11):3819-30. DOI: 10.1021/ja909524e
Source: PubMed


In recent years, the biological sciences have seen a surge in the development of methods, including high-throughput global methods, for the quantitative measurement of biomolecule levels (i.e., RNA, proteins, metabolites) from cells and tissues. Just as important as quantitation of biomolecules has been the creation of approaches that uncover the regulatory and signaling connections between biomolecules. Our specific interest is in understanding peptide metabolism in a physiological setting, and this has led us to develop a multidisciplinary approach that integrates genetics, analytical chemistry, synthetic chemistry, biochemistry, and chemical biology to identify the substrates of peptidases in vivo. To accomplish this we utilize a liquid chromatography-mass spectrometry (LC-MS)-based peptidomics platform to measure changes in the peptidome as a function of peptidase activity. Previous analysis of mice lacking the enzyme dipeptidyl peptidase 4 (DPP4(-/-) mice), a biomedically relevant peptidase, using this approach identified a handful of novel endogenous DPP4 substrates. Here, we utilize these substrates and tissues from DPP4(-/-) mice to improve the coverage of the peptidomics platform by optimizing the key steps in the workflow, and in doing so, discover over 70 renal DPP4 substrates (up from 7 at the beginning of our optimization), a 10-fold improvement in our coverage. The sequences of these DPP4 peptide substrates support a broad role for DPP4 in proline-containing peptide catabolism and strengthen a biochemical model that interlinks aminopeptidase and DPP4 activities. Moreover, the improved peptidome coverage also led to the detection of greater numbers of known bioactive peptides (e.g., peptide hormones) during the analysis of gut samples, suggesting additional uses for this optimized workflow. Together these results strengthen our ability to identify endogenous peptide substrates through improved peptidome coverage and demonstrate a broader potential of this peptidomics platform.

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Available from: Arthur D Tinoco, Aug 22, 2015
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    • "Tissue peptide isolation and fractionation were previously described [24], [36], [37]. Briefly, frozen spinal cords were placed in 500 µL of water and boiled for 10 minutes to inactivate any residual proteolytic activity prior to tissue homogenization. "
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    ABSTRACT: Substance P (SP) is a prototypical neuropeptide with roles in pain and inflammation. Numerous mechanisms regulate endogenous SP levels, including the differential expression of SP mRNA and the controlled secretion of SP from neurons. Proteolysis has long been suspected to regulate extracellular SP concentrations but data in support of this hypothesis is scarce. Here, we provide evidence that proteolysis controls SP levels in the spinal cord. Using peptidomics to detect and quantify endogenous SP fragments, we identify the primary SP cleavage site as the C-terminal side of the ninth residue of SP. If blocking this pathway increases SP levels, then proteolysis controls SP concentration. We performed a targeted chemical screen using spinal cord lysates as a proxy for the endogenous metabolic environment and identified GM6001 (galardin, ilomastat) as a potent inhibitor of the SP 1-9-producing activity present in the tissue. Administration of GM6001 to mice results in a greater-than-three-fold increase in the spinal cord levels of SP, which validates the hypothesis that proteolysis controls physiological SP levels.
    PLoS ONE 07/2013; 8(7):e68638. DOI:10.1371/journal.pone.0068638 · 3.23 Impact Factor
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    • "Chymotrypsinogens are highly expressed in the exocrine pancreas, but expression in β-cells has also been shown (25), which is confirmed in this study and in INS1E cells. Interestingly, in a study in mice, chymotrypsin has been identified as a target for DPP-4 (21). A peptide fragment (VPAIQPVLTG) containing the DPP-4 H2N-Xaa-Pro consensus motif was found at a 10-fold higher level in gut tissue from DPP-4−/− versus DPP-4+/+ mice. "
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    ABSTRACT: The incretin hormone glucagon-like-peptide 1 (GLP-1) promotes glucose homeostasis and enhances beta-cell function. GLP-1 receptor agonists (GLP-1 RA) and dipeptidyl peptidase-4 (DPP-4) inhibitors, which inhibit the physiological inactivation of endogenous GLP-1, are used for the treatment of type 2 diabetes. Using the Metabochip we identified three novel genetic loci with large effects (30-40%) on GLP-1 stimulated insulin secretion during hyperglycemic clamps in non-diabetic Caucasian individuals (TMEM114; CHST3 and CTRB1/2; n=232, all p≤8.8*10(-7)). rs7202877 near CTRB1/2, a known diabetes risk locus, also associated with an absolute 0.51±0.16% (5.6±1.7 mmol/mol) lower A1C response to DPP-4 inhibitor treatment in G allele carriers but there was no effect on GLP-1 RA treatment in type 2 diabetes patients (n=527). Furthermore, in pancreatic tissue we show that rs7202877 acts as expression quantitative trait locus for CTRB1 and CTRB2, encoding chymotrypsinogen, and increases fecal chymotrypsin activity in healthy carriers. Chymotrypsin is one of the most abundant digestive enzymes in the gut where it cleaves food proteins into smaller peptide fragments.Our data identify chymotrypsin in the regulation of the incretin pathway, development of diabetes and response to DPP-4 inhibitor treatment.
    Diabetes 05/2013; 62(9). DOI:10.2337/db13-0227 · 8.10 Impact Factor
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    • "In particular, because of the modern multidisciplinary approaches , in recent years we have assisted to an improved peptidome and lipidome coverage [6] [7] [8]. Endogenous peptides (PT)/proteins and lipids (LP) represent two important classes of biomolecules owing to their multiple biological roles in physiological and in pathological conditions. "
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    ABSTRACT: Herein we report the use of mesoporous aluminosilicate (MPAS) for the simultaneous extraction of peptides and lipids from complex body fluids such as human plasma and synovial fluid. We show that MPAS particles, given their mesostructural features with nanometric pore size and high surface area, are an efficient device for simultaneous extraction of peptidome and lipidome from as little as a few microliters of body fluids. The peptides and the lipids, selected and enriched by MPAS particles and rapidly visualized by MALDI-TOF MS, could form part of a diagnostic profile of the "peptidome" and the "lipidome" of healthy versus diseased subjects in comparative studies. The ability of this approach to rapidly reveal the overall pattern of changes in both lipidome and peptidome signatures of complex biofluids could be of valuable interest for handling large numbers of samples required in -omics studies for the purpose of finding novel biomarkers.
    Proteomics 11/2012; 12(22). DOI:10.1002/pmic.201200204 · 3.81 Impact Factor
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